Authors: Yan Weiling, Liao Rongfeng, Li Ning
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Objective
To investigate the relationship between p53 and miR-29b in myofibroblasts (MFs) induced from human Tenon fibroblasts (HTFs).
Methods
Human Tenon tissues were obtained from strabismus surgery patients in the First Affiliated Hospital of Anhui Medical University from March to July 2016.The primary HTFs were cultured by tissue culture method and passaged to 2-4 generations.The cells were divided into 4 groups: HTFs group cells were normally cultured, the TGF-β1 activation group cells was activated with 10 ng/ml tumor necrosis factorβ1(TGF-β1), the siRNA transfection group and the negative control group were transfected with p53 siRNA and negative siRNA, respectively.The expression of p53 protein and cell migration ability of HTFs and MFs were detected by immunohistochemical and cell scratch assay.The relative expression level of miR-29b and p53 mRNA in each group was detected by real-time PCR.This study followed the Helsinki declaration and the patients or their guardians signed informed consent.
Results
The primary HTFs had long fusiform shape, and were arranged in a spiral shape.After activated by TGF-β1, the cells were transformed into MFs, and the cell morphology was irregular and the arrangement was disordered.The staining intensity of p53 protein in TGF-β1 activation group was significantly stronger than that in HTFs group(t=-10.384, P<0.05). The migration distance of TGF-β1 activation group cells was significantly larger than that of HTFs group (P<0.05). The relative expression level of p53 mRNA was 1.00±0.00, 3.95±2.61, 0.06±0.06 and 0.98±0.16 in HTFs group, TGF-β1 activation group, siRNA transfection group and negative control group, respectively; the relative expression levels of miR-29b was 1.00±0.00, 0.54±0.09, 5.10±2.31 and 1.03±0.09 in HTFs group, TGF-β1 activation group, siRNA transfection group and negative control group, respectively; the overall differences were statistically significant (p53 mRNA: F=6.688, P=0.006; miR-29b: F=13.640, P=0.000). The relative expression of p53 mRNA was significantly higher and the relative expression of miR-29b was significantly lower in the TGFβ1 activation groups than those in the HTFs group and siRNA transfection group, the differences were statistically significant (all at P<0.05).
Conclusions
During the process of HTFs transforming to MFs, the expression of p53 is upregulated and the expression of miR-29b is downregulated, Supressing the expression of p53 can upregulate the expression of miR-29b.There may exist interactions between p53 and miR-29b, which regulate the formation of fibrotic scars.